Field of the Invention
The present invention relates to electromagnetic emissions from fluorescence, chemiluminescence, bioluminescence, and luminescence molecule, and more specifically, to detection of such emissions in wavelengths from UV-visible to near IR and at a directional or fixed angle.
Background of Related Technology
Surface plasmon fluorescence spectroscopy (SPFS),1 a technique that utilizes the interactions of fluorescent species with thin metal films, is becoming a useful tool in the analytical biosciences. In SPFS, fluorescent species typically attached to biomolecules that are brought within close proximity to the metal surface via biorecognition events between metal surface bound biomolecules and the fluorescently-labeled biomolecules as part of the bioassays constructed on the metal surface. The fluorescence emission detected from the sample side (free-space emission) or through the prism (surface plasmon coupled fluorescence, SPCF) is then used to quantify the biomolecule of interest. In this regard, attomolar sensitivity in immunoassays based on SPFS has been reported.2 One can also find other reports on SPSF for DNA hybridization3-5 and protein detection.6 
In SPFS, two modes of excitation of the fluorescent species can be achieved: 1) Kretschmann (KR) configuration: through a prism, 2) reverse Kretschmann (RK) configuration: directly from the air or sample side.1 The description of both modes of excitation has been given elsewhere.7 In fluorescence-based biosensing applications that utilize optically dense medium, such as whole blood, the KR configuration is typically considered for the excitation of fluorescent species. This is due to the effective excitation of fluorescent species by the excitation light in the form of an evanescent wave which penetrates several hundred nanometers into the optically dense medium from the surface of the metal. On the other hand, in RK configuration, the efficiency of excitation of the fluorescent species in optically dense medium can be considerably less than as compared to KR due to the sample thickness and inner filter effect. Regardless of the excitation mode, the fluorescence emission can be detected as both free-space isotropic emission and/or highly directional SPCF emission. One can visually see the SPCF emission as a cone or as a “ring” from the back of the film when a hemispherical prism is employed.
The choice of metal in SPSF is usually gold1, 8 since it is inert and amenable to chemical modification without the loss of physical and electronic properties. Despite their versatility, the use of gold thin films is limited to the visible spectral range. In recent years, there has been a resurgence in the investigation of other metals to alleviate this problem: silver9 and aluminum10 and zinc thin films7 were shown the work in the UV and UV to visible spectral range, respectively. It was also shown that copper thin films11 can also be used with fluorophores emitting >550 nm. It is important to also note that the angle of reflectivity minimum varies with the type and thickness of the metal used due to the optical properties of the metal. In this regard, Fresnel calculations have been shown to be an excellent tool for prediction of the interaction of light with metals.
Near-infrared fluorescence is attractive in many settings such as in optical imaging and immunoassays, because it circumvents some of the problems associated with fluorescence in the visible region. However, the detection limits and sensitivity are still limited by the photostability and quantum yield of the near IR-fluorophore (label) and therefore still remain a primary concern in fluorescence spectroscopy and imaging today. Thus, it would be advantageous to develop a system that provides strong signals with increased photostability for a near IR-fluorophore. As such, there is a continued search for metal(s) that can function in a broad wavelength range with the possibility of covering the wavelengths for many commercially available fluorophores. That is, a single metal thin film can utilize fluorophores from the UV to NIR without the need to change metal.
Chemiluminescence is a very useful analytical technology for the quantitative detection of biomolecules of interest. Chemiluminescence emission, which is a result of chemical reactions between an organic dye and an oxidizing agent in the presence of a catalyst, is the primary tool in this technology. Chemiluminescence emission occurs as the energy from the excited states of organic dyes, which are chemically induced, decays to ground state. The duration and the intensity of the chemiluminescence emission are mostly dependent on the extent of the chemical reagents present in the reaction solution. Despite the usefulness of the chemiluminescence technology, chemiluminescence emission is isotropic and the efficiency of detection of chemiluminescence emission by current optical detectors is very low. In this regard, the chemiluminescence technology still requires much needed improvement in the efficiency of detection of chemiluminescence emission.
Thus it would be advantageous to overcome the shortcomings discussed above and it would be ideal to develop a system to wherein the reflectivity minimum for such a metal thin film would occur at a fixed wavelength, which would alleviate the need to change the observation angle, which is one of the disadvantages of using silver, gold, copper and/or zinc thin films.